Lesions of the biliary system in Atlantic salmon (Salmo salar L.)

1Norwegian Institute of Food Fisheries and Aquaculture Research (Nofima), Tromsø, Norway 2Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Ås, Norway 3Cargill Aqua Nutrition, Bergen, Norway 4Cermaq Group AS, Oslo, Norway 5Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Norwegian University of Life Sciences, Ås, Norway

Gallbladders with mucosal outpouchings (diverticula) were found in a population of Atlantic salmon farmed in sea water. Soft tissue calcification affected the skin, skeletal muscle and gut, as identified by histological observations. In addition, inflammation of muscle and intestinal tissue and lymphocytic and fibrous reaction were present in the liver. The general fish population was characterized by poor growth and unspecified mortalities. Here we describe the observed lesions and discuss them in the light of the general health status of the population.

| S TUDY P OPUL ATION
This study was part of a feeding trial in Finnmark county, northern Norway. The fish used were 147 g Atlantic salmon smolts transferred to six commercial seawater cages in November 2018 (68,415 salmon per cage) and harvested in May 2020 (2.9 kg). The fish were fed according to nutritional requirements with either a constant or seasonal adjusted EPA + DHA regimes in triplicates.
In October 2019 (11 months at sea) and May 2020 (at harvest), fish were randomly selected from each of the sea cages (n = 10) for biometric determinations. Tissue samples from four fish per sea cage waere collected from pyloric caeca, distal intestine and liver (liver only from May sampling), while skin with attached red and white skeletal muscle was collected from five fish per sea cage. In May, gallbladders with visual pathological changes were collected from four individuals. Tissue samples were fixed in 10% phosphate-buffered formalin and stained with haematoxylin and eosin (H&E) according to Li et al. (2019), Alcian blue and PAS (AB/ PAS) according to Sveen et al. (2021) and Von Kossa according to Culling et al. (2014). For registered pathologies, the reader is referred to Appendix S1.

| RE SULTS
After 17.5 months in sea, growth was poorer than expected with 2.9 kg average harvest weight, whereas the expected slaughter weight for Atlantic salmon is 4-5 kg (Barrett et al., 2022). The cumulative mortality for the production was 13.3% with the weekly number of mortalities per cause given in Figure 1

F I G U R E 4 Calcification of skin and muscle tissue. (a-c) Calcified lesions (black arrow) were found in skin (Ski) dermis, myosepta (Myo), red and white muscle tissue (Mu). (d-f) Red and white muscle tissue with inflammation (white arrow) and vacuolization (pattern filled arrow).
Tissue section (a-c) stained with Von Kossa, (d-f) with AB/PAS dermis-, myosepta-and muscle tissue were affected, accompanied by inflammation and vacuolization of red muscle tissue (Figure 4). In May 2020, 70% of the skin samples, 38% of distal intestine and 8% of the pyloric caeca samples had calcified lesions (Figures 3 and 4), while only one fish had inflammation in red muscle tissue.

| DISCUSS ION
Here we present findings from a population of A. salmon, which upon harvest was characterized by poor growth, unspecified mortalities, incidences of calcinosis and lesions of the digestive and biliary system ( Figure 5). The recorded causes of mortality and the average mortality rate represent the general trend in the industry (Sommerset et al., 2020). The large size of the diverticula suggests that this condition must have persisted for some time. The nature of the calcified foci and fibrosis of the hepatic duct further suggests a chronic state of the lesions. Such a chronic disease progression likely reduces the fitness of the animals and overlaps well in time with the unspecified mortalities recorded for this production. The high incidences of lesions in the gut and the biliary system likely affected nutrient digestion and absorption negatively, which may have contributed to the poor growth observed for the population.
The lesions of the gallbladders resemble that of intramural diverticulosis described in humans (Mark & Melnick, 1964;Ross et al., 1955). Ross et al. describe these diverticula as a likely consequence of chronic inflammation and increased luminar pressure, resulting in pulsion diverticula into the loose stroma of the gallbladder wall. Here, the high percentage of lesions in the biliary system and gut may indicate that these events are connected. Bile acids are further vital for the absorption of dietary fat and fat-soluble vitamins such as Vitamin D. Dysregulation of vitamin D is related to calcification processes (Black & Kanat, 1985); hence, it is plausible that the biliary lesions may be connected to the calcified foci. Unfortunately, liver/gut and skin/gallbladder samples were collected from different individuals, and this hypothesis needs further elucidation.
The samples for this study were collected from random fish at a commercial production site; hence, we believe that the findings are representative for the population. The series of lesions may represent a health problem which may have been overlooked previously.

ACK N OWLED G EM ENTS
The authors express their gratitude to Mira Hansen at the Veterinary Institute laboratory at Harstad for excellent histological processing of tissue samples.

This work was funded by the project 'Omega-3 for salmon under
Arctic conditions -optimization of use and effects on fish health and fillet quality' which was supported by Researcsh Council of Norway grant no 296296 -RFF ARKTIS and the industrial partners Cermaq and Cargill.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest associated with this publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Lene R. Sveen
https://orcid.org/0000-0003-3394-3462 F I G U R E 5 Pathological findings at harvest. The percentage of lesions is given as (n of lesions/n of samples analysed) separately for each tissue. The percentage of nodular gall bladders is given by (n observations/total number of fish sampled at harvest).